Irina Tikhanovich

Forkhead box class O transcription factors in liver function and disease.

The forkhead box transcription factor class O (FOXO) family represents a group of transcription factors that is required for a number of stress-related transcriptional programs including antioxidant response, gluconeogenesis, cell cycle control, apoptosis, and autophagy. The liver utilizes several FOXO-dependent pathways to adapt to its routine cycles of feeding and fasting and to respond to the stresses induced by disease. FOXO1 is a direct transcriptional regulator of gluconeogenesis, reciprocally regulated by insulin, and has profound effects on hepatic lipid metabolism. FOXO3 is required for antioxidant responses and autophagy and is altered in hepatitis C infection and fatty liver. Emerging evidence suggests dysregulation of FOXO3 in some hepatocellular carcinomas. FOXOs are notable for the extensive number of functionally significant posttranslational modifications that they undergo. Recent advances in our understanding how FOXOs are regulated are providing a more detailed picture of how specific combinations of posttranslational modifications alter both nuclear translocation as well as transcriptional specificity under different conditions. This review summarizes emerging knowledge of FOXO function in the liver, FOXO changes in liver disease, and the posttranslational modifications responsible for these effects.

Regulation of FOXO3 by phosphorylation and methylation in hepatitis C virus infection and alcohol exposure.

Hepatitis C virus (HCV) infection produces chronic liver injury that is significantly exacerbated by alcohol consumption. While multiple mechanisms contribute to this synergy, a viral‐induced loss of antioxidant responses has been shown to play an important role. This study examined the effects of HCV infection and alcohol on the regulation of the transcription factor FOXO3, an important regulator of Mn‐superoxide dismutase (SOD2) expression, a tumor suppressor, and a component of the hepatic antioxidant response system. FOXO3 was activated by either HCV or alcohol alone but suppressed by the combination. To understand this paradoxical result, we applied a capillary isoelectric focusing (IEF) method to determine the pattern of FOXO3 posttranslational modifications (PTMs) induced by HCV and alcohol. We observed the presence of multiple different nuclear and cytosolic species of FOXO3 and used antiphosphoserine, acetyl‐lysine, methylarginine, and ubiquitin antibodies to identify the PTM patterns present in each species. HCV caused multiple changes including phosphorylation of FOXO3 at S‐574, a novel c‐Jun N‐terminal kinase (JNK) site, which promoted nuclear translocation and transcription. Ethanol suppressed arginine‐methylation of FOXO3 promoting nuclear export and degradation of the JNK phosphorylated form. Human liver biopsy samples showed the presence of the HCV‐specific form of FOXO3 in HCV‐infected livers but not in normal liver or nonalcoholic steatohepatitis. Conclusion: The development of this novel IEF method for the simultaneous quantification of differently modified FOXO3 species allowed us to demonstrate how HCV and alcohol combine to modify a complex pattern of FOXO3 PTMs that contribute to pathogenesis. This approach will allow further dissection of the role of protein PTMs in viral liver disease. (Hepatology 2014;58:58–70)

Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling

Background: Innate immune signaling requires multiple mechanisms to suppress signaling in the absence of stimulation. Results: TNF receptor associated factor 6 (TRAF6) activity is regulated by reversible arginine methylation. Conclusion: Arginine methylation of TRAF6 inhibits signaling in the absence of Toll-like receptor ligands. Significance: Reversible TRAF6 methylation is a novel mechanism that controls innate immune responses. Arginine methylation is a common post-translational modification, but its role in regulating protein function is poorly understood. This study demonstrates that, TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase involved in innate immune signaling, is regulated by reversible arginine methylation in a range of primary and cultured cells. Under basal conditions, TRAF6 is methylated by the methyltransferase PRMT1, and this inhibits its ubiquitin ligase activity, reducing activation of toll-like receptor signaling. In response to toll-like receptor ligands, TRAF6 is demethylated by the Jumonji domain protein JMJD6. Demethylation is required for maximal activation of NF-κB. Loss of JMJD6 leads to reduced response, and loss of PRMT1 leads to basal pathway activation with subsequent desensitization to ligands. In human primary cells, variations in the PRMT1/JMJD6 ratio significantly correlate with TRAF6 methylation, basal activation of NF-κB, and magnitude of response to LPS. Reversible arginine methylation of TRAF6 by the opposing effects of PRMT1 and JMJD6 is, therefore, a novel mechanism for regulation of innate immune pathways.

Hepatitis C and alcohol exacerbate liver injury by suppression of FOXO3.

Hepatitis C virus (HCV) infection exacerbates alcoholic liver injury by mechanisms that include enhanced oxidative stress. The forkhead box transcription factor FOXO3 is an important component of the antioxidant stress response that can be altered by HCV. To test whether FOXO3 is protective for alcoholic liver injury, we fed alcohol to FOXO3(-/-) mice. After 3 weeks, one third of these mice developed severe hepatic steatosis, neutrophilic infiltration, and >10-fold alanine aminotransferase (ALT) elevations. In cell culture, either alcohol or HCV infection alone increased FOXO3 transcriptional activity and expression of target genes, but the combination of HCV and alcohol together caused loss of nuclear FOXO3 and decreased its transcriptional activity. This was accompanied by increased phosphorylation of FOXO3. Mice expressing HCV structural proteins on a background of reduced expression of superoxide dismutase 2 (SOD2; Sod2(+/-)) also had increased liver sensitivity to alcohol, with elevated ALT, steatosis, and lobular inflammation. Elevated ALT was associated with an alcohol-induced decrease in SOD2 and redistribution of FOXO3 to the cytosol. These results demonstrate that FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcohol-induced liver injury.

Protein arginine methyltransferase 1 modulates innate immune responses through regulation of peroxisome proliferator-activated receptor γ-dependent macrophage differentiation

Arginine methylation is a common posttranslational modification that has been shown to regulate both gene expression and extranuclear signaling events. We recently reported defects in protein arginine methyltransferase 1 (PRMT1) activity and arginine methylation in the livers of cirrhosis patients with a history of recurrent infections. To examine the role of PRMT1 in innate immune responses in vivo, we created a cell type-specific knock-out mouse model. We showed that myeloid-specific PRMT1 knock-out mice demonstrate higher proinflammatory cytokine production and a lower survival rate after cecal ligation and puncture. We found that this defect is because of defective peroxisome proliferator-activated receptor γ (PPARγ)-dependent M2 macrophage differentiation. PPARγ is one of the key transcription factors regulating macrophage polarization toward a more anti-inflammatory and pro-resolving phenotype. We found that PRMT1 knock-out macrophages failed to up-regulate PPARγ expression in response to IL4 treatment resulting in 4-fold lower PPARγ expression in knock-out cells than in wild-type cells. Detailed study of the mechanism revealed that PRMT1 regulates PPARγ gene expression through histone H4R3me2a methylation at the PPARγ promoter. Supplementing with PPARγ agonists rosiglitazone and GW1929 was sufficient to restore M2 differentiation in vivo and in vitro and abrogated the difference in survival between wild-type and PRMT1 knock-out mice. Taken together these data suggest that PRMT1-dependent regulation of macrophage PPARγ expression contributes to the infection susceptibility in PRMT1 knock-out mice.

Demethylase JMJD6 as a New Regulator of Interferon Signaling: Effects of HCV and Ethanol Metabolism

Background & Aims Alcohol-induced progression of hepatitis C virus (HCV) infection is related to dysfunction of innate immunity in hepatocytes. Endogenously produced interferon (IFN)α induces activation of interferon-stimulated genes (ISGs) via triggering of the Janus kinase–signal transducer and activator of transcription 1 (STAT1) pathway. This activation requires protein methyltransferase 1–regulated arginine methylation of STAT1. Here, we aimed to study whether STAT1 methylation also depended on the levels of demethylase jumonji domain-containing 6 protein (JMJD6) and whether ethanol and HCV affect JMJD6 expression in hepatocytes. Methods Huh7.5-CYP (RLW) cells and hepatocytes were exposed to acetaldehyde-generating system (AGS) and 50 mmol/L ethanol, respectively. JMJD6 messenger RNA and protein expression were measured by real-time polymerase chain reaction and Western blot. IFNα-activated cells either overexpressing JMJD6 or with knocked-down JMJD6 expression were tested for STAT1 methylation, ISG activation, and HCV RNA. In vivo studies have been performed on C57Bl/6 mice (expressing HCV structural proteins or not) or chimeric mice with humanized livers fed control or ethanol diets. Results AGS exposure to cells up-regulated JMJD6 expression in RLW cells. These results were corroborated by ethanol treatment of primary hepatocytes. The promethylating agent betaine reversed the effects of AGS/ethanol. Similar results were obtained in vivo, when mice were fed control/ethanol with and without betaine supplementation. Overexpression of JMJD6 suppressed STAT1 methylation, IFNα-induced ISG activation, and increased HCV-RNA levels. In contrast, JMJD6 silencing enhanced STAT1 methylation, ISG stimulation by IFNα, and attenuated HCV-RNA expression in Huh7.5 cells. Conclusions We conclude that arginine methylation of STAT1 is suppressed by JMJD6. Both HCV and acetaldehyde increase JMJD6 levels, thereby impairing STAT1 methylation and innate immunity protection in hepatocytes exposed to the virus and/or alcohol.

Arginine methylation regulates c-Myc-dependent transcription by altering promoter recruitment of the acetyltransferase p300

Protein arginine methyltransferase 1 (PRMT1) is an essential enzyme controlling about 85% of the total cellular arginine methylation in proteins. We have shown previously that PRMT1 is an important regulator of innate immune responses and that it is required for M2 macrophage differentiation. c-Myc is a transcription factor that is critical in regulating cell proliferation and also regulates the M2 transcriptional program in macrophages. Here, we sought to determine whether c-Myc in myeloid cells is regulated by PRMT1-dependent arginine methylation. We found that PRMT1 activity was necessary for c-Myc binding to the acetyltransferase p300. PRMT1 inhibition decreased p300 recruitment to c-Myc target promoters and increased histone deacetylase 1 (HDAC1) recruitment, thereby decreasing transcription at these sites. Moreover, PRMT1 inhibition blocked c-Myc–mediated induction of several of its target genes, including peroxisome proliferator-activated receptor γ (PPARG) and mannose receptor C-type 1 (MRC1), suggesting that PRMT1 is necessary for c-Myc function in M2 macrophage differentiation. Of note, in primary human blood monocytes, p300–c-Myc binding was strongly correlated with PRMT1 expression, and in liver sections, PRMT1, c-Myc, and M2 macrophage levels were strongly correlated with each other. Both PRMT1 levels and M2 macrophage numbers were significantly lower in livers from individuals with a history of spontaneous bacterial peritonitis, known to have defective cellular immunity. In conclusion, our findings demonstrate that PRMT1 is an important regulator of c-Myc function in myeloid cells. PRMT1 loss in individuals with cirrhosis may contribute to their immune defects.

FOXO Transcription Factors in Liver Function and Disease

The forkhead box transcription factor class O (FOXO) family represents a group of transcription factors that is required for a number of stress-related transcriptional programs including antioxidant response, gluconeogenesis, cell cycle control, apoptosis, and autophagy. The liver utilizes several FOXO-dependent pathways to adapt to its routine cycles of feeding and fasting and to respond to the stresses induced by disease. FOXO1 is a direct transcriptional regulator of gluconeogenesis, reciprocally regulated by insulin, and has profound effects on hepatic lipid metabolism. FOXO3 is required for antioxidant responses and autophagy and is altered in hepatitis C infection and fatty liver. Emerging evidence suggests dysregulation of FOXO3 in some hepatocellular carcinomas. FOXOs are notable for the extensive number of functionally significant posttranslational modifications that they undergo. Recent advances in our understanding how FOXOs are regulated are providing a more detailed picture of how specific combinations of posttranslational modifications alter both nuclear translocation as well as transcriptional specificity under different conditions. This review summarizes emerging knowledge of FOXO function in the liver, FOXO changes in liver disease, and the posttranslational modifications responsible for these effects.

PRMT1 and JMJD6 dependent arginine methylation regulate HNF4α expression and hepatocyte proliferation

Alcohol is a well‐established risk factor for hepatocellular carcinoma (HCC), but the mechanisms by which it promotes liver cancer are not well understood. Several studies have shown that cellular protein arginine methylation is inhibited by alcohol. Arginine methylation is controlled by the reciprocal activity of protein arginine methyltransferases, primarily protein arginine methyl transferase 1 (PRMT1), and a demethylase Jumonji C domain‐containing protein 6 (JMJD6). The aim of this study was to explore the role of arginine methylation changes in alcohol pathogenesis. We found that PRMT1 activity is inhibited in livers of mice fed with alcohol compared to pair‐fed mice. Using hepatocyte‐specific PRMT1 knockout mice, we identified that loss of PRMT1 results in enhanced hepatocyte proliferation and a 33% increase in liver size. This increased hepatocyte proliferation was associated with reduced expression of hepatocyte nuclear factor 4 alpha (Hnf4α), an important regulator of liver tumorigenesis. We found that PRMT1 regulates Hnf4α expression directly through arginine methylation at the (Hnf4α) promoter. In the absence of PRMT1, JMJD6 can demethylate the Hnf4α promoter and suppress its expression. We were able to restore Hnf4α expression and abolish the increase in hepatocyte proliferation by knockdown of JMJD6 in PRMT1 knockout mice. Knockdown of JMJD6 in alcohol‐fed mice similarly increased Hnf4α expression. We then examined whether loss of arginine methylation might play a role in alcohol‐associated liver cancers. We examined 25 human HCC specimens and found a strong correlation (R = 0.8; P < 0.01) between arginine methylation levels and Hnf4α expression in these specimens, suggesting that the above mechanism is relevant in patients. Conclusion: Taken together, these data suggest that PRMT1 inhibition, such as induced by alcohol, may result in epigenetic changes leading to loss of Hnf4α. This effect may contribute to alcohols ability to promote liver tumors. (Hepatology 2018;67:1109–1126)

Retinoids: The Link Between Alcohol and Interferon?

One of the greatest accomplishments in the history of the field of hepatology is the 25year effort that led from the discovery of the hepatitis C virus (HCV) to the understanding of its life cycle and ultimately the development of highly effective targeted therapy. Perhaps less well appreciated is the extent to which the global scientific effort against HCV has transformed our knowledge of liver disease, host-viral interactions, and antiviral immune responses. Among these insights are the understanding of the reversibility of cirrhosis, the prognostic interpretation of liver histology, and the genesis of hepatocellular carcinoma. However, of all the scientific fruits of HCV research, none have been as significant as the profound insights gained into the nature of the antiviral interferon (IFN) pathway and the factors that determine its efficacy in different individuals. Although the development of new anti-HCV drugs has reached a state of maturity, basic scientific understandings of the nature of the livervirus interaction continue to emerge. One of the central issues that remain unresolved is the relationship between HCV and alcohol consumption. It is well documented that HCV and alcohol each exacerbate liver injury caused by the other, but even more profound is the observation that alcohol consumption leads to a more than seven-fold increase in the rate of virological failure of IFN treatment. In spite of these strong clinical data, the mechanisms responsible for this effect are poorly understood—and their relevance, if any, to IFN-free treatment regimens is unknown. Interferons generated by hepatocytes in response to viral infection both clear virus from infected cells and make uninfected cells resistant to infection. In the case of HCV, the viral RNA is recognized by the pattern receptor and RNA helicase, RIG-I, and this triggers a signaling cascade involving the adapter protein MAVS, the downstream kinases TBK1 and IKKe, and the transcription factors IRF3 and IRF7. In hepatocytes, this results in the secretion of IFN-b. This process engages the effector arm of the IFN pathway where type 1 IFN binding to the receptors INFAR1 and IFNAR2 results in activation of the receptor-bound kinases TYK2 and JAK1. These in turn cause tyrosine phosphorylation of STAT1 and STAT2, which then form a heterotrimeric complex of pSTAT1, pSTAT2, and IRF9. This IFN-stimulated gene factor 3 is the active transcription factor that binds many IFN-sensitive response elements (ISREs) inducing IFN-stimulated genes (ISGs) that suppress viral replication by multiple mechanisms. Prior to the development of direct-activing antivirals, HCV therapy was almost wholly based on trying to activate this endogenous method to clear the virus. Endogenous IFN responses still determine the outcome of acute infections and may even play some role in direct-activing antiviral efficacy. Initially, there was little understanding of how alcohol affected IFN responses, but as replicon and in vitro infectious virus models of HCV became available, multiple effects were observed. On one hand, lipid peroxidation, as caused by alcohol, is a potent inhibitor of HCV replication through direct effects on viral proteins. On the other hand, alcohol also suppresses the IFN pathway by reducing STAT1 tyrosine phosphorylation, suppressing permissive arginine methylation of STAT1, decreasing expression of STAT1 and STAT2, and inducing the accumulation of two Abbreviations: ADH, aldehyde dehydrogenase; HCV, hepatitis C virus; IFN, interferon; ISG, interferon-stimulated gene; ISRE, interferon-sensitive response element; RARE, retinoic acid responsive.